From Big Data to Relevant data: Ribo-seq and its applications
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From Big Data to Relevant data: Ribo-seq and its applicationsPasha Baranov "NGS Data after the Gold Rush“ Norwich, UK, May 2014
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Genetics: up to 2.4 higher chance than general population.
What to sequence?Lung cancer
80-90% of lung cancer cases attributed to smoking.
Median age of lung cancer occurrence in US is 70 years.
DNA
RNA
Protein
RNA-seq
?
Ribosomal profiling (ribo-seq)Ingolia et al (2009) Science 324: 218-23
Lysis, RNA digestion
Ribosomal profiling (ribo-seq)
Lysis, RNA digestion
Density gradient orcushion separation
Ingolia et al (2009) Science 324: 218-23
Ribosomal profiling (ribo-seq)
Lysis, RNA digestion
Density gradient orcushion separation
Adaptor ligation RT PCR Circularization
Decircularization
Sequencing
Alignment to a reference sequence
Differential gene expression analysis
Ingolia et al (2009) Science 324: 218-23
mRNA level is only approximation of protein synthesis
Immediate response to oxidative stress (1 hour)
Andreev et al 2014 submitted
How do you know what is translated?
CTGGAAGAAGTAAACGCCGAGCTGGAACAGCCGGATGTCTGGAACGAACC G R S K R R A G T A G C L E R T Frame 1 L E E V N A E L E Q P D V W N E P Frame 2 W K K * T P S W N S R M S G T N P Frame 3
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicityCAGCTAGTGCGTGCTGTC123123123123123123
Triplet periodicity
N+1
N+2
N+3
Switches between reading frames can be seen on ribosome profiles
Human Antizyme 1 mRNA
Michel et al 2012 Genome Res 22:2219-2229
uORFs & nORFs
Michel et al 2012 Genome Res 22:2219-2229
GWIPS-viz
http://gwips.ucc.ie
AcknowledgmentsGWIPS-viz team:Audrey Michel Gearoid Fox Patrick O'Connor Stephen Heaphy Paddy Mullan Claire DonohueRomika SainiZena Abbas
Oxidative stress work:Dmitry Andreev Patrick O’ConnorMoscow State University:Terenin, Dmitriev, ShatskyTrinity College Dublin: Kenny, Fahey, Cormican, Morris
Dual coding:Audrey MichelUniversity College Cork:Choudhury, AtkinsBerkley: IngoliaCambridge: Firth
Financial resources: